With the advent of the use of flow cytometry instrumentation in clinical and research environments, it has become concurrently necessary for purposes of toxicology, diagnosis, the identification of preferred hybridoma cell lines and the like to be able to discriminate between cell types in a heterogeneous cell population. Historically this has been accomplished by either employing stains specific for viable cells as opposed to nonviable cells, i.e. the vital type stains, or by the measurement of multiple parameters such as size, degree of fluorescence exhibited and the like in an effort to distinguish between the effects of a stain applied generally to all the cells in the mixture.
For instance, in an article by Dr. Myron R. Melamed entitled "Cytotoxic Test Automation: A Live Dead Cell Differential Counter" (Science, 163:285-286, 1969) a method was described whereby a cell spectrofluorometer was employed for the automatic discrimination and enumeration of live and dead cells in a cytotoxic test. The vital indicator employed was trypan blue because nonviable cells take up trypan blue and hence become stained while living cells exclude the dye and thus remain unstained. Since trypan blue effectively absorbs light, a measurement of absorption can be correlated to the percentage of stained or nonviable cells versus unstained viable cells.
It is an object of the present invention to provide a method which does not rely on the viability of cells and a resultant differential uptake of a vital type stain but rather is capable of discriminating between viable cells by use of a non-absorbence measurement technique.
Differentiation of cells based on different dye uptakes and measurements of scatter and absorption has been described by Friedman et al. in U.S. Pat. No. 3,785,735. Specifically illustrated in that patent is the differentiation of live and dead cells based upon the different uptakes of a vital stain such as trypan blue. It was found that dead cells which were stained by trypan blue exhibited characteristics of high absorbence and low scatter whereas live cells exhibited low absorbence and high scatter characteristics. Friedman measured absorbence by placing the detectors at a wide angle to the incident illuminating light beam in an effort to reduce noise signals apparently greater with direct (zero angle) absorbence measurement. Friedman relies on absorbence and scatter measurements in order to discriminate between live and dead cells, he does not teach how differentiation between different, live cell types may be accomplished without reliance on absorbence measurements.
It is an object of the present invention to provide a method whereby cells stained with an absorbing surface stain and unstained cells present in a heterogeneous mixture may be differentiated by reliance on low angle and wide angle scatter measurements but without reliance on additional absorbence measurements. In fact, scatter and absorbence may be shown to be inversely related in that the more light that is absorbed by a surface, the less light is available for scatter. Thus, Friedman claims that dead cells (stained by trypan blue) absorb more light than unstained cells and thereby implies a characteristically lower scatter measurement with a stained cell than with an unstained cell. It has been discovered that in fact, the wide angle measurement of a cell stained with an absorbing type stain demonstrates increased scatter over that produced by unstained cells. It is consequently an object of the present invention to utilize this discovery in discriminating between stained and unstained cells.
The use of an absorbing dye to detect cytoplasmic immumoenzyme staining of a viral antigen has been described by Leary et al, J. Histochem. Cytochem. 24, 1249 (1976). Cells containing virus were fixed in formaldehyde and then reacted with an antibody against the virus. A second antibody conjugated to the enzyme peroxidase and reactive with the first antibody was then reacted with the cells. When the substrate diaminobenzidine dihydrochloride was then reacted with the cells, a brown reaction product was deposited in those cells with the peroxidase enzyme, and hence those cells containing antibody against the virus. The presence of brown reaction product was detected by analyzing the cells on an ORTHO CYTOFLUOROGRAF* Flow Cytometer with an argon ion laser. On the CYTOFLUOROGRAF axial light loss, angular range 0.degree.-1.degree. relative to the laser beam, (called "low angle scatter" by Leary et al) and forward light scatter, angular range 1.degree.-19.degree. relative to the laser beam, (called "wide angle scatter" by Leary et al) were measured. Reactive cells showed less forward light scatter and more axial light loss than unreactive cells. FNT *Trademark
A similar effect is reported for light-absorbing stain on the surface of cells in Ortho Protocol Number 8, published by and available from Ortho Diagnostic Systems Inc. In this protocol, "Cytographic Analysis of Con-A Receptor Site in Plasmacytoma Cells," a method is described for immunoperoxidase staining of concanavalin A on the surface of cells. The staining results in a light-absorbing red stain on the surface of the cells. Axial light loss and forward light scatter from a helium neon laser were measured on a CYTOGRAF 6300A*. Stained cells showed markedly decreased forward light scatter, while axial light loss did not change greatly. FNT *Trademark
An object of the present invention is to replace the relatively insensitive axial light loss measurement with a measurement of light scattered at very wide angles (approximately 55.degree. to 125.degree. as measured on the CYTOFLUOROGRAF). Wide angle light scatter has been found by us to be significantly affected by the presence of light-absorbing stains in or on the surface of cells.
Other conventional methods of cell differentiation have been based on the measurement of different fluorescent characteristics of cells uniformly stained with a single type of fluorochrome. For example, U.S. Pat. No. 3,684,377 to Adams et al. describes a method of analyzing living white blood cells from other blood components utilizing various compositions comprising, in part, acridine orange as the fluorochrome. The cells are subjected to blue laser illumination and detection of green fluorescence provides a distinguishing feature between white blood cells and other blood components. Combination of this information with the detection of red fluorescence emitted from individual white cells, permits discrimination between white cell types. It is an object of the present invention to provide a method which does not depend upon the detection of multiple fluorescent characteristics in order to effectuate cell type differentiation.
Differentiation between blood components has also been accomplished by two dimensional signal analysis of a detected scatter signal as the cells passed through a focused collimated light source. Such a method is described in U.S. Pat. No. 4,202,625 to Weiner et al. which describes methods and apparatus whereby blood components, specifically red blood cells and platelets, may be distinguished. Weiner, however, does not teach how scatter measurements in conjunction with absorbence dyes may be used to differentiate cell types. It is an object of the present invention to provide such methodology.